Research on pebrine (also called silkworm microsporidiosis) started with the pebrine epidemic all over France in 1845. Louis Pasteur associated the “particle” that he observed as the causative factor of pebrine, and then Balbiani identified it as Nosema bombycis (J. V. Maddox et al., 2000). There are two routes of infection for Nosema bombycis, i.e. horizontal transmission and vertical transmission, wherein the vertical transmission causes huge harm to silkworm eggs production in sericulture and makes considerable negative effects on yield and quality of cocoon in silkworm cocoon production at the same time, which seriously affected the development of downstream economy in the silk industry chain (S. F. Cai, et al., 2011). Subsequent to the nationwide outspreading of pebrine in France, Nosema bombycis has always been a significant detection item in silkworm eggs production in various countries as well as import and export trade. In late 19th century, the period that sericulture in Japan was most developed, “Female Moth Microscopic Examination Regulations” and “Silkworm Disease Prevention Regulations” were listed into the Japanese Constitution (Takeshi Kawarabata, 2003). In China, pebrine has been listed as Class II diseases in quarantine of imported and exported animals (List of Infectious and Parasitic Diseases of Imported Animals for Class I and Class II of P.R.C., 1992, Agri. (Quarantine) No. 12) as well.
At first, people distinguished Nosema bombycis mainly according to the clinical characters of pebrine by visual inspection. After microscope was invented, people detected Nosema bombycis according to its morphology and size by microscopic examination, which enhanced the sensitivity and efficiency of detection to a certain extent, and thus the pebrine pandemic in France was restrained and the worldwide sericulture was rescued. However, there are some obvious drawbacks in microscopic examination, such as the high demands for skills and experiences of operating personnel, and Nosema bombycis being so tiny that it is difficult to distinguish microsporidium and its analogues due to low specificity and sensitivity of the detection method through general microscopic examination, and especially, Nosema bombycis in silkworm egg is generally immature spores which are hardly identified and determined by general optical microscope.
Following the development of PCR (Polymerase Chain Reaction) technology, PCR methods have been used for detecting Nosema bombycis, which reaches a higher sensitivity. “Molecular clock” is an effective measure for analysis of biological system evolution on the molecular level, and SSU rRNA (16S rDNA) is a commonly used “molecular clock” in the researches on microorganism evolution (Pei A. Y., et al., 2010). Most of the target genes, at which the primers designed in the researches of PCR detection technology for detecting pebrine aim, are SSU rRNA, but there are very few reports about the primers aiming at other microsporidium genes as much less primers are designed or with low sensitivity. PCR primers V1f/530r, designed by Baker et al. (1995) and Terry et al. (1999) according to the highly conserved sequences of SSU rRNA of closely related species of microsporidium, may identify specific bands of DNA templates in various species of microsporidiums with amplification about 450 bp, but without specificity to Nosema bombycis. Also present inventors found that the sensitivity of detection was extremely low when using the above-mentioned primers to detect microsporidium in silkworm eggs, which implied that a suppression factor might exist in silkworm egg extract and interfered with the PCR amplification of Nosema bombycis (N.b) DNA. Microsporidium is parasitic in silkworm egg, but the content of the egg is apparently higher than that of the microsporidium to be detected. DNAs of both silkworm eggs and microsporidium exist in the extracted DNA sample, so that silkworm eggs DNAs seriously interfere with the detection of the microsporidium DNAs. Thus, higher requirements are needed for direct detection of microsporidium when using silkworm egg DNA as a template.
Besides, in practical works, particularly in quarantine works, detection of various pathogens of microsporidium in samples is so important that no omission or false detection is allowed. Thus, it is desirable to have a primer set for universal detection of various microsporidiums with excellent detective sensitivity, which is also highly sensitive when using silkworm egg DNA as a template, and such primer set is valuable in extensive applications and of significance in practical detection works of microsporidium.